IL-27 Inhibits Anti-Mycobacterium tuberculosis Innate Immune Activity of Primary Human Macrophages

Mycobacterium tuberculosis (M tuberculosis) is an intracellular pathogen that primarily infects macrophages. Despite a robust anti-mycobacterial response, many times macrophages are unable to control M. tuberculosis. The purpose of this study was to investigate the mechanism by which the immunoregulatory cytokine IL-27 inhibits the anti-mycobacterial activity of primary human macrophages. We found concerted production of IL-27 and anti-mycobacterial cytokines by M. tuberculosis-infected macrophages in a toll-like receptor (TLR) dependent manner. Notably, IL-27 suppressed the production of anti-mycobacterial cytokines TNFα, IL-6, IL-1β, and IL-15 by M. tuberculosis-infected macrophages. IL-27 limits the anti-mycobacterial activity of macrophages by reducing Cyp27B, cathelicidin (LL-37), LC3B lipidation, and increasing IL-10 production. Furthermore, neutralizing both IL-27 and IL-10 increased the expression of proteins involved in LC3-associated phagocytosis (LAP) pathway for bacterial clearance, namely vacuolar-ATPase, NOX2, and RUN-domain containing protein RUBCN. These results implicate IL-27 is a prominent cytokine that impedes M. tuberculosis clearance.


Introduction
Mycobacterium tuberculosis (M. tuberculosis), the causative agent for tuberculosis is a highly successful intracellular pathogen making it one of the leading infectious causes of mortality worldwide 1 . M. tuberculosis resides in the macrophages and manipulates macrophage-induced host cell defense mechanisms such as antigen presentation, phagosomal maturation, cytokine production, and antimicrobial pathways for its own survival 2,3 . The engagement of surface Toll-like receptors (TLR)-2 and -4 on macrophages with its cognate M. tuberculosis pathogen-associated molecular pattern (PAMP) activates signaling and phagolysosome pathways important for the production of cytokine/chemokines and M. tuberculosis degradation, respectively [4][5][6][7] . TLR-2 and -4 mediate the release of cytokines TNFα, IL-6, IL-1β, IL-12 by macrophages in response to M. tuberculosis infection, which is critical for host resistance to infection due to their ability to activate cathelicidin (hCAP) and defensin mediated direct antimicrobial mechanisms [8][9][10][11][12] However, despite strong anti-M. tuberculosis innate immune mechanisms a substantial proportion of individuals infected with M. tuberculosis are unable to control the infection.
IL-27 is a heterodimeric cytokine of the IL-12 family, composed of p28 and Epstein-Barr virus-induced protein 3 (EBI3) subunits that are structurally similar to the p35 and p40 subunits of IL-12, respectively 13 . Activated macrophages and dendritic cells secrete IL-27, which regulates both innate and adaptive immune responses. Unlike IL-12, IL-27 inhibits anti-M. tuberculosis T cell effector functions, and phagosomal acidification and compromises control of M. tuberculosis growth by macrophages [14][15][16][17] An increased level of IL-27 has been reported during M. tuberculosis infection and treatment with IL-12 along with IL-27 neutralization restricts the growth of M. tuberculosis 15,18 . We recently reported that neutralizing IL-27 controls replication of M. tuberculosis in the settings of HIV-M. tuberculosis co-infection 19 . The underlying mechanism by which IL-27 impedes anti-mycobactericidal activity of macrophages remains less defined.
The purpose of the present study was to examine the effect of IL-27 on host defense of primary human macrophages in vitro infected with M. tuberculosis. We present evidence that IL-27 is produced by human macrophages in TLR dependent manner along with other anti-M. tuberculosis cytokines. We further show that IL-27 inhibits proinflammatory cytokine production in response to infection with M. tuberculosis, and subsequent mycobactericidal activity of macrophages. These results provide an insight into the immune therapeutic approaches that may be adopted to control M. tuberculosis infection.

Cell isolation and generation of primary human macrophages
Peripheral blood mononuclear cells (PBMC) were isolated from freshly obtained blood by Ficoll density centrifugation (GE Healthcare) and used for the generation of primary monocyte-derived macrophages (hereafter macrophages). Monocytes were isolated by plate adherence method, and subsequently cultured with RPMI1640 supplemented with AB human serum and M-CSF (10 ng/ml) for 7-days. Media was replaced every third day with fresh culture media supplemented with M-CSF.

M. tuberculosis culture
M. tuberculosis Erdman, provided by Dr. Fred Quinn (University of Georgia, Athens), was maintained in Middlebrook 7H9 broth containing albumin, dextrose, catalase (ADC) as previously described 20 . For macrophages infections, bacteria were plated on Middlebrook 7H10 agar containing 10% oleic acid-albumin-dextrose-catalase (OADC). All assays involving M. tuberculosis were performed in Biosafety Level-3 following the institutional biosafety guidelines.

Macrophage infection and treatment
Macrophages (0.3 x 10 6 / well) were plated in 12-well plates in antibiotic-free RPMI 1640 medium and 10% human serum. Cells were infected with Erdman at a multiplicity of infection (MOI) of 1:5 for 3 hrs; subsequently, cells were washed and treated with Gentamycin Sulfate (30 μg/ml; VWR Life Sciences) for additional 2 hours to kill extracellular bacteria, and cultured in RPMI 1640 with 10% human serum. For some experiments, macrophages were incubated with blocking anti-TLR2 or -TLR4 or isotypematched control antibodies (10 μg/ml) (all from Thermo Scientific) for 30 minutes at 37°C followed by infection with M. tuberculosis. For some experiments, M. tuberculosis-infected macrophages were cultured with recombinant (r) -IL-27 (10 or 15 ng/ml), neutralizing IL-27 or isotype matched control antibodies (all from R&D systems), IL-10 (Thermo Scientific) antibodies ( The derivation and characterization of these strains has previously been described Culture supernatants or cellular lysates were prepared at 24-hours post-infection and stored at -80°C until further use. To determine the effect of protein phosphatase 2ac (PP2ac), M. tuberculosis-infected macrophages were cultured with PP2Ac agonist forskolin (FK) or antagonist okadaic acid (OA) (both from Tocris), intracellular replication or expression of proteins was measured in the cellular lysates at 24-hours post-infection.

Measurement of cell cytotoxicity
Cell cytotoxicity of macrophages infected with M. tuberculosis and treated with rIL-27, or anti-IL-27 and IL-10 antibodies was determined using CyQUANT™ LDH cytotoxicity assay kit according to manufacturer's instructions (Supplementary Figure 1). Briefly, 50 μl of culture supernatant was added to equal volume of reaction mixture and incubated at room temperature for 30 minutes, followed by addition of stop solution. Absorbance was measured at 490nm and 680 nm as reference wavelength. Cells treated with lysis buffer, and untreated media served as maximum LDH and spontaneous LDH activity controls, respectively; % cytotoxicity = [Treatment LDH activity -spontaneous LDH activity / maximum LDH activity -spontaneous LDH activity] x 100. Data is presented as percentage viability = 100 -% cytotoxicity.

Measurement of intracellular M. tuberculosis
Macrophages were infected with M. tuberculosis as detailed above. Cells were lysed with 0.1% SDS at day-0 and day -3 post-infection, and cellular lysates were serially diluted and plated in triplicate on Middlebrook 7H10 agar supplemented with OADC enrichment. The number of colonies was counted after 3 weeks and colony-forming units (CFU) / ml was determined.

Fluorescence microscopy
Macrophages were infected with M. tuberculosis as detailed above. Cells were fixed for 30 minutes using formaldehyde, then permeablized with 0.2% Triton X-100 for 10 minutes, and incubated with 2% bovine serum albumin to block non-specific binding of antibodies. Cells were incubated with anti-human-p62-Alexa Fluor 647 or -NOX2-APC/Cy7 at 1:100 dilution overnight at 4°C, followed by mounting with Prolong Gold antifade agent with nuclear stain DAPI. Cells were imaged on Nikon A1R confocal microscope using 75X objective.

Quantitative Reverse-Transcription Polymerase chain reaction (qRT-PCR)
qRT-PCR was performed as previously described 19 . Total RNA was isolated from macrophages using TRIzol™ reagent (Thermo Fisher Scientific) according to the manufacturer's protocol; 500 ng RNA was used for cDNA synthesis using Superscript II reverse transcriptase (Thermo Fisher Scientific). SyBrGreen master mix (Thermo Fisher Scientific) was used in a 10μL reaction volume that included 10 ng of cDNA for primer pairs (1 μM) specific for Human Cytochrome P450 oxidase (Cyp7B1; forward CACCCGACACGGAGACCTT, reverse TCAACAGCGTGGACACAAACA). The Human acidic ribosomal protein (HuPO; forward CATTCTATCATCAACGGGTACAA, reverse AGCAAGTGGGAAGGTGTAATCC) was used as a housekeeping gene. Data were analyzed to calculate the relative quantification of the Cyp27B1 gene in comparison to the HuPO gene by comparative Ct method (2-ΔCt) 23 .
The quantity of hCAP was measured in the culture supernatants at 24 hrs post-infection using hCAP LL-37 kit (Hycult Biotech).

Statistical analysis
Data are expressed as mean values ± standard error mean (SEM). Paired Student's t-tests were used to determine the statistical significance for in vitro experiments. Statistical analysis was performed using Graphpad Prism 8 (La Jolla, CA). P-values of <0.05 were considered statistically significant.

Concurrent production of IL-27 and proinflammatory cytokines by human macrophages
M. tuberculosis-infected macrophages elicit cytokine production that plays a critical role in deciding the outcome of M. tuberculosis infection. Among these TNF-α, IL-6 and IL-1β are critical for an optimal innate immune-mediated control of M. tuberculosis.
Initially, we characterized our primary human macrophage system by flow cytometry. Our primary human macrophages expressed CX3CR1 similar to lung "tissue-resident" macrophages and consisted of CD11b + HLA DR + CD169 + CD206 + macrophages (alveolar macrophages) and HLA DR + CD14 + CD36 + macrophages (interstitial macrophages) ( Figure  1A), thus reflecting the similarities with lung resident cells [24][25][26] . Subsequently, we infected primary human macrophages with M. tuberculosis; similar to previous studies we found M. tuberculosis-infected macrophages produced pro-inflammatory cytokines as early as 3-hours post-infection ( Figure 1B-D). IL-27 is an immune-regulatory cytokine that has both proinflammatory and anti-inflammatory properties, and its manipulation influences proinflammatory cytokine production by macrophages. We found human macrophages also produce IL-27 upon infection with M. tuberculosis ( Figure 1E and Supplementary Figure 2).
Signaling pathways activated because of engagement of TLRs with its cognate M. tuberculosis pathogen-associated molecular pattern (PAMP) are important for cytokine production by macrophages 2,4,5 . In this regard, we found that blocking TLR-2 or Gollnick  -4 prior to infection with M. tuberculosis decreased the production of TNF-α and IL-6 ( Figure 1F-G). Of note, similar to TNF-α and IL-6, the quantity of IL-27 decreased when TLR-2 or TLR-4 were blocked prior to infection ( Figure 1I and Supplementary Figure 2 Figure 1J). Collectively these studies demonstrate that M. tuberculosis-induced TLR signaling not only activates macrophages to produce anti-M. tuberculosis cytokines, but also induces immunoregulatory cytokines such as IL-27 in a TLR dependent manner. These studies show that IL-27 inhibits anti-mycobacterial cytokine production, and augments intracellular replication of M. tuberculosis.

IL-27 inhibits the mycobactericidal activity of human macrophages
We found IL-27 inhibits M. tuberculosis-induced IL-15 production (31 ± 2 vs 22 ± 2.1 pg/ml; p= 0.0003) ( Figure 2I). IL-15 is important to maintain memory T cells, and for the induction of Cyp27B1 and the downstream antimicrobial peptide hCAP critical for the anti-mycobacterial activity of macrophages 10,27 Figure 3A).
It has previously been demonstrated that hCAP mediates the anti-mycobacterial activity of macrophages by upregulation of the autophagy pathway, which results in the formation of autophagosomes 9, 28 . In the next set of experiments, we sought to study if IL-27 interferes with the autophagy pathway of macrophages infected with M. tuberculosis. During autophagy, cytosolic microtubule-associated protein 1 light chain 3B (LC3B)-I is converted to LC3B-II (LC3B lipidation); increased expression of LC3B-II is an indicator of autophagy induction, or its accumulation due to the inhibition of autophagic flux (degradation of polyubiquitin-binding protein p62 (sequestosome 1). Initially, we studied autophagy in macrophages infected with M. tuberculosis. Consistent with previous findings, we found infection with M. tuberculosis increased LC3B lipidation but inhibited p62 degradation.
Importantly, treatment with rapamycin induced autophagic flux (Supplementary Figure 3B).  Figure 3F and H). Immunofluorescence also showed few p62 puncta in M. tuberculosis infected macrophages treated with rapamycin, the addition of rIL27 did not affect rapamycin induced p62 puncta ( Figure 3I). A previous study demonstrated that inhibition of serine/threonine protein phosphatase-2Ac (PP2Ac) increased LC3B-II and decreased p62 expression in M. bovis infected mouse macrophages.
We found that the inhibition in the expression of LC3B-II by IL-27 was independent  Figure 4). Of note, treatment with PP2Ac inhibitor OA decreased intracellular replication of M. tuberculosis (Supplementary Figure 4B). Collectively, these findings suggest that IL-27 inhibits the anti-mycobacterial activity of macrophages by inhibiting autophagy induction.

IL-27 regulates the expression of proteins involved in LC3 associated phagocytosis (LAP)
Our findings so far suggest that IL-27 augments intracellular M. tuberculosis replication, and inhibits the anti-mycobacterial activity of macrophages. IL-27 driven inhibition of anti-mycobacterial activity is dependent on LC3B-II but does not affect autophagic flux.
In the next experiments, we sought to investigate the anti-mycobacterial pathway inhibited by IL-27. It is well established that IL-27 inhibits phagosomal acidification by blocking vacuolar ATPases (V-ATPase) 15 The Immunosuppressive cytokine IL-10 is produced by macrophages in response to M. tuberculosis infection and inhibits phagolysosomal mediated control of mycobacteria 31,32 .

Discussion
Macrophages are central to innate immune-mediated control of M. tuberculosis. Activation of TLR on human macrophages induces proinflammatory cytokine production, which augments lysosomal activity for bacterial clearance. Prior studies have shown that M. tuberculosis hijacks antimicrobial machinery of macrophages for its own survival [33][34][35]  Europe PMC Funders Author Manuscripts that IL-27 inhibits hCAP and interferes with LC3 associated phagocytosis (LAP) pathway, which ultimately inhibit the expression of V-ATPase critical for bacterial clearance.
TLRs 2 and 4 are the major pathogen recognition receptors (PRR) for M. tuberculosis.
The concurrent induction of IL-10, IL-4, and TGF-β by mycobacterial molecules such as lipoarabinomannan, phosphatidylinositol mannoside, triacylated or diacylated lipoproteins inhibit IFN-γ signaling in macrophages, allowing M. tuberculosis to evade host immune response 39 . In this regard, the recently identified immunoregulatory cytokine IL-27 opposes IL-12 mediated M. tuberculosis clearance 16, 17 . We found IL-27 was increased in response to M. tuberculosis infection in our system. However, the quantity of IL-27 secreted is much less than other cytokines. We did find significant expression of IL27p28 in the cellular lysates (Supplementary Figure 2) suggesting that: (i) even a small quantity of secreted IL-27 is sufficient to transmit biological function to neighboring cells, and (ii) IL-27 retained inside macrophages inhibits anti-mycobacterial signals, impeding mycobacterial control. In mice, IL27p28 (p28) is induced both in IFN-γ independent and dependent manner by utilizing distinct pathways under the two conditions [40][41][42] . IFN-γ independent p28 production is dependent on TLR-4/myeloid differentiation factor 88 (MyD88)-NF-kB c-Rel pathway; whereas in response to IFNγ, p28 is produced in an interferon regulatory factor -1 (IRF1)-dependent manner 40

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts hydroxylase (Cyp27b1) and subsequent bioconversion of circulating 25-hydroxyvitamin D3 into bioactive 1,25D 3 leading to the induction of hCAP 10,27 . The commercial serum we used to culture macrophages contained natural 25(OH) D3 at a range previously shown to be sufficient to trigger hCAP expression 27 . Therefore, the suppression of Cyp27b1 and subsequent LL-37 ( Figure 3A and B) by IL-27 could be a direct effect of IL-15 inhibition by IL-27 ( Figure 2I). One pathway that links LL-37 to mycobacterial degradation is the activation of autophagy, which results in autophagosome formation and their subsequent fusion with lysosomes 9, 28 . Notably virulent M. tuberculosis has developed strategies that impair autophagy at the step of autophagic flux and autophagosome-lysosome fusion 34,35 . IFN-γ treatment of macrophages overcomes autophagy blockade in M. tuberculosisinfected cells 10,11 . Consistent with previous studies we found, M. tuberculosis infection IL-27 allowed for V-ATPase recovery 16 . We and others have shown that IL-27 in myeloid cells signals through STAT3, a transcription factor that mediates anti-inflammatory effect 14,19,45 . STAT3 inhibition of human macrophages by small-molecule niclosamide increases lysosomal acidification and its association with BCG 46,47 . The anti-inflammatory cytokine IL-10 also signals through STAT3 and prevents acidification of mycobacteria containing phagosomes 31,32,48,49 . Thus, both IL-27 and IL-10 operate together or in a similar manner to oppose, phagosomal acidification and anti-mycobacterial immune response of macrophages. Kalliolias et al showed that IL-27 priming of macrophages suppresses IL-10 gene expression and protein production in response to TLR synthetic ligands. In this study, we provide the first evidence that IL-27 produced during M. tuberculosis infection regulates IL-10 production, and neutralizing both of these cytokines profoundly affects the anti-mycobacterial activity of human macrophages. Future studies utilizing animal models of tuberculosis will address the effect of IL-27 neutralization on granuloma, cellular recruitment to the lung microenvironment, secondary lymph nodes, and containment of M. tuberculosis in vivo. Nevertheless, our studies on primary human macrophages suggest that regulating the cytokine environment is an important approach to establish the antimycobacterial state of macrophages.
Previous studies also establish that autophagy-mediated clearance of M. tuberculosis by macrophage requires IFN-γ induced activation signals 9-11, 28, 52, 53 . During M. tuberculosis infection, lymphocytes are the primary cells producing IFN-γ, which activate macrophages in a paracrine manner [54][55][56][57][58] . IFN-γ gene expression and protein production by lymphocytes in M. tuberculosis-infected mice is detectable as early as 10-days and 14-days post-infection [59][60][61] . Our in vitro model without exogenous IFN-γ resembles an early infection time point when the effect of activation signals on macrophages generated by other immune cells is minimum. ROS produced by host enzymes is also critical for controlling M. tuberculosis both in vitro, in experimental disease models, and in patients suffering from chronic granulomatous disease [62][63][64][65][66] . Although regulation of ROS production during M. tuberculosis infection is not well-defined, it is dependent on the multimeric enzyme NOX2 in TLR dependent manner, where NOX2 aggregates and activates oligomeric protein complexes to participate in the host immune defense [67][68][69] . LC3 associated phagocytosis (LAP) is another phagolysosomal pathway that is characterized by the association of LC3 with phagosomal membrane 30,70 and is active in M. tuberculosis infected cells 76 . Consistent to LAP activation pathway, our immunoblotting, gene expression and microscopy studies demonstrate induction of NOX2 gp91 phox ( Figure 4C and F, and Supplementary Figure 5A) and p47 phox (Supplementary Figure 5B)

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts